A safe, effective method of dissolving vascular thrombi is urgently needed, owing to the life-threatening complications of thromboembolic disease. The most common of such disorders is the formation of thrombi, in a blood vessel or heart cavity that remain at the point of formation. Thrombi in heart vessels, for example, can restrict blood flow, resulting in myocardial infarction. Coronary arteriographic studies indicate that 87% of transmural myocardial infarctions are caused by occlusive coronary artery thrombosis. DeWood et al., N. Eng. J. Med. 303:397-902 (1980).
In addition, parts of a thrombus may dislodge from its point of attachment and move through the blood vessels until it reaches a point where passage is restricted. The resulting blockage is known as a thromboembolism.
Plasminogen activators function mainly by converting the zymogen plasminogen into plasmin, which is regarded as the end stage non-specific proteolytic enzyme in fibrinolysis. Plasmin lyses the fibrin matrix and acts on other components of the thrombus as well. The efficiency of the lytic activity of exogenous plasminogen activator depends significantly on the amount of plasmin it generates from endogenous plasminogen, which is present in free-form in circulating blood and bound form on the fibrin surfaces of thrombi and hemostatic plugs. In any thrombolytic therapy, it is desirable to generate the maximum amount of plasmin on the fibrin surface i.e., plasmin immobilized on the thrombus, with a minimum quantity of plasmin (if possible, none at all) in the circulating blood. While immobilized plasmin efficiently carries out the lysis of the thrombus and helps establish reperfusion, circulating plasmin exhibits fibrinogenolytic activity, degrades platelet receptors, digests thrombospondin, and consequently decreases the platelet aggregation capacity of the patient. The result is the so called "lytic state", which is accompanied by various undesired complications.
A variety of plasminogen activators have been utilized to promote dissolution of thrombi and restoration of blood circulation to blocked vessels. However, the available plasminogen activators are far from ideal. Streptokinase, which is of bacterial origin, is antigenic. A loading dose is needed to neutralize pre-existing antistreptokinase antibodies. Streptokinase treatment must be limited in duration because of an increase in such antibody levels. Conard et al., Semin. Throm. Haemostas. 13:212-222 (1987). Streptokinase works efficiently to dissolve thrombi if administered locally, but local administration requires catheterization, which may often preclude administration promptly enough to provide significant benefit to the patient. Systemic administration of streptokinase in acute myocardial infarction causes an undesirable side effect by degrading fibrinogen and severely depleting plasminogen in the circulation. Mentzer et al., Am. J. Cardiol. 57:1220-1226 (1986).
Another widely used plasminogen activator, urokinase, is available in three forms. So called "two-chain urokinase" (hereinafter "urokinase") can be extracted from human urine or prepared from kidney cell cultures. Another form, "single-chain urokinase" or "pro-urokinase" (hereinafter "scu-PA"), a precursor of urokinase, has been isolated from human fluids and also obtained by recombinant DNA technology. A low molecular weight form of urokinase is obtained from long plasmin digestion of two-chain urokinase.
Although urokinase is not antigenic, its mechanism of plasminogen activation parallels that of streptokinase, resulting in a significant fibrinogenolytic response in the circulating blood. Single-chain urokinase has promising characteristics. However, it has not yet been adequately tested in man. In a small study of normal human subjects, scu-PA displayed fibrinolytic activity comparable to urokinase, and a lower systemic action than urokinase. Trubestein et al., Hemostasis 17:238-244 (1987).
Non-urokinase plasminogen activators, which include tissue plasminogen activator ("t-PA"), are physiologic activators of plasminogen which may be manufactured by cell culture and genetic engineering. t-PA is capable of binding fibrin, and is believed to convert plasminogen to plasmin at the fibrin surface. Thus, it works more efficiently than streptokinase or urokinase when administered systemically. However, initial clinical trials indicate that lysis of fibrinogen is still significant during administration of t-PA. Sherry, N. Engl. J. Med. 313:1014-1017 (1985).
Although it has high specificity for fibrin, t-PA has certain limitations. It has a very short biological half-life time in the circulation of humans, on the order of five minutes. Cellular clearance of t-PA by the liver is believed responsible for this limited biological half-life time. Rapid decrease of t-PA in the blood after intravenous administration constitutes a major difficulty in drug therapy with respect to maintaining an adequate concentration of the activator.
Plasma contains plasminogen activator inhibitors which can bind and inactivate plasminogen activators. Continuous infusion of large doses of t-PA are therefore necessary to overcome the effect of these inhibitors. At physiologic levels, free t-PA is bound by the plasma inhibitor, PAI-1. Wun et al., Blood 69:1354-1362 (1987). The latter has shown to be present in elevated levels in the plasma of patients with myocardial infarction, Hamsten et al., N. Engl. J. Med. 313:1557-1563 (1985); recurrent deep venous thrombosis, deJong et al., Thromb. Haemostas. 57:140-143 (1987); and coronary artery disease, Paramo et al., Br. J. Med. 291:573-574 (1985). When purified t-PA is added to plasma to create higher-than physiologic levels to overcome the inhibitory effect of PAI-1, it complexes with other inhibitors present in plasma, such as C1-inhibitor, alpha.sub.2 -antiplasmin and macroglobulin. Thorsen et al., Biochem. Biophys. Acta 802:111-118 (1984); Rijken et al., J. Lab. Clin. Med. 101:285-294 (1983); Kruithoff et al., Blood 64:907-913 (1984).
Administration of urokinase, streptokinase and t-PA results in an increased tendency for bleeding, limiting the usefulness of these agents, particularly during surgical procedures.
Streptokinase and urokinase do not have inherent fibrin specificity. They indiscriminately convert both circulating and fibrin-surface bound plasminogen into plasmin at presently-used therapeutic dosages. While t-PA and scu-PA have fibrin specificity, they have short half-life times in the blood. A high dosage must be infused over a long period of time in order to maintain a therapeutic concentration in the blood. Consequently, hemostatic imbalances are encountered. Moreover, since the specificities of t-PA and scu-PA for fibrin are not absolute, they leave a small amount of activators in the liquid phase, and some amount of plasmin is gradually produced in the circulating blood.
Complexes have been formed from urokinase and antifibrin murine monoclonal antibodies. Bode et al., Science 229:765-767 (1985); Bode et al., J. Biol. Chem. 262:10819-10823 (1987) and J. Mol. Cell Cardiol. 19:335-341 (1987). In an in vitro model of a thrombus in a circulating plasma loop, the antibody-urokinase complex displayed a greater rate of fibrinolysis than an equivalent amount of pure urokinase. However, therapeutic use of such complexes is unattractive due to the immunogenicity of murine monoclonal antibodies.
U.S. Pat. No. 4,564,596 discloses urokinase derivatives comprising urokinase covalently bonded to fibrinogen, either directly or through a specific aliphatic diamine. The derivatives are reported to have an increased affinity for fibrin and prolonged fibrinolytic effect. However, fibrinogen has procoagulant action, and may promote further development of thrombi. Thrombus-bound fibrinogen which is carrying urokinase will promote extension of the thrombus via either platelet aggregation or fibrin deposition, acting locally as a procoagulant agent. This may lead to reocclusion of the vessel.
Fibrinogen must be treated with thrombin, in order to maximize targeting to thrombi. Thus, the fibrinogen-urokinasae hybrid of U.S. Pat. No. 4,564,596 requires the presence of a thrombotic state for optimal performance. Moreover, fibrinogen is an adhesive protein, since it contains the amino acid sequence Arg-Gly-Asp. Fibrinogen binds to a variety of cells, providing a bridge between cells. Consequently, much of the injected fibrinogen-urokinase hybrid may bind to cells in the circulating blood, making less hybrid available to bind to thrombi.
Human fibrin fragment E.sub.1 binds specifically to polymers of fibrin, Olexa et al., Proc. Natl. Acad. Sci. USA 77:1374-1378 (1980), and has the ability to bind to aged as well as fresh thrombi. Knight et al., J. Clin. Invest. 72:2007-2013 (1983). .sup.123 I-labeled fragment E.sub.1 has been used to detect venous thrombi in patients. Knight et al., Radiology 156:509-514 (1985). In addition to fragment E.sub.1, fragment E.sub.2 and peptides having an amino acid sequence intermediate between fragments E.sub.1 and E.sub.2 may be labeled to detect thrombi, as disclosed in U.S. Pat. No. 4,427,646.
CNBr-digest fragments of fibrinogen have been shown to accelerate the activation of t-PA. Nieuwenhuizen et al, Biochem. Biophys. Acta 755:531-533 (1983); Lijnen et al, Eur. J. Biochem. 144:541-544 (1984); Nieuwenhuizen et al, Biochem. Biophys. Acta 748:86-92 (1983). However the fibrinogen fragments afford no protection from inactivation of t-PA by plasminogen activator inhibitors present in plasma.
What is needed is a thrombus-dissolving agent having more potent lytic activity than the known plasminogen activators, but without the disadvantages of such known activators. Ideally, a thrombolytic agent should have very high fibrin specificity, and should immobilize itself on or near the fibrin surface of a thrombus. It should activate only fibrin surface-bound plasminogen without activating circulating plasminogen. In addition, a lytic agent should have a very high potential for plasmin generation on a thrombus surface, and have a prolonged half-life time in the circulation so that lengthy, high-dose infusions are not necessary. The thrombolytic agent which has the potential to produce more plasmin from a given amount of plasminogen will perform as a better lytic agent, because the required amount of plasmin concentration on the thrombus surface can be achieved by using a smaller amount of activator units.
By "plasminogen activator" is meant any agent capable of activating the zymogen plasminogen to the fibrinolytic enzyme plasmin.
By "fibrin fragment" is meant any fragment resulting from the single or sequential plasmin cleavage of intact cross-linked or non-cross-linked fibrin ("native fibrin fragment"), and any such fragment which is further cleaved, derivatized or modified in any manner, while retaining a substantial portion of the native fragment amino acid sequence ("non-native fibrin fragment").
By the term "linked", as used herein in describing the union between a plasminogen activator and a fibrin fragment, is meant any form of chemical association or bond, including, but not limited to non-covalent complex formation, covalent bonding (including but not limited to covalent bonding by means of one or more cross-linking agents), and the like. Also included in the scope of such associations is the formation of a unitary protein by genetic engineering, resulting from the co-expression of genetic information for all or part of the fibrin fragment and plasminogen activator as a single protein.